Isolation of antisense oligonucleotides

ABSTRACT

The present invention relates to a method of isolating fully thioated single stranded antisense oligonucleotides from a biological solution, which method comprises the steps of contacting the biological solution with an immobilised metal ion adsorption chromatography (IMAC) resin to adsorb the antisense oligonucleotides to the resin and subsequently contacting the resin with an eluent under conditions that provide desorption of the antisense oligonucleotides from the resin, wherein the fully thioated antisense oligonucleotides are separated from incorrectly thioated antisense oligonucleotides in the solution. The invention also relates to the use of an immobilised metal ion adsorption chromatography (IMAC) resin for isolation of fully thioated single stranded antisense oligonucleotides from a biological solution.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. § 371 and claims priorityto international patent application number PCT/SE2003/001784 filed Nov.17, 2003, published on Jun. 10, 2004 as WO 2004/048569 and also claimspriority to patent application number 0203521-0 filed in Sweden on Nov.28, 2002; the disclosures of which are incorporated herein by referencein their entireties.

TECHNICAL FIELD

The present invention relates to a method of isolating antisenseoligonucleotides from other components of a biological solution.

BACKGROUND

Biotechnological methods are used to an increasing extent in theproduction of proteins, peptides, nucleic acids and other biologicalcompounds, for research purposes as well as in order to prepare novelkinds of drugs. Due to its versatility and sensitivity to the compounds,chromatography is often the preferred purification method in thiscontext. The term chromatography embraces a family of closely relatedseparation methods, which are all based on the principle that twomutually immiscible phases are brought into contact. More specifically,the target compound is introduced into a mobile phase, which iscontacted with a stationary phase. The target compound will then undergoa series of interactions between the stationary and mobile phases as itis being carried through the system by the mobile phase. Theinteractions exploit differences in the physical or chemical propertiesof the components in the sample.

Interactions between a target compound and metal chelating groupspresent on the stationary phase are utilised in a chromatographicpurification method denoted immobilised metal ion adsorptionchromatography (IMAC), also known as metal chelating affinitychromatography (MCAC), which is often used for the purification ofproteins. The principle behind IMAC lies in the fact that manytransition metal ions can coordinate to phosphate groups and nitrogenatoms, such as in the amino acids histidine, cystein, and tryptophan,via electron donor groups on the amino acid side chains. To utilise thisinteraction for chromatographic purposes, the metal ion must beimmobilised onto an insoluble support. This can be done by attaching achelating group to the chromatographic matrix. Most importantly, to beuseful, the metal of choice must have a higher affinity for the matrixthan for the compounds to be purified. Examples of suitable coordinatingions are Cu(II), Zn(II), Ni(II), Ca(II), Co(II), Mg(II), Fe(III),Al(III), Ga(IIl), Sc(III) etc.

Various chelating groups are known for use in IMAC, such asiminodiacetic acid (IDA), which is a tridentate chelator, andnitrilotriacetic acid (NTA), which is a tetradentate chelator. Elutionof an IMAC resin is as regards proteins commonly performed by additionof imidazol. Alternatively, elution is conventionally performed bylowering the pH.

In recent years, IMAC has successfully been used for the purification ofproteins and peptides, wherein His-tags have been introduced byrecombinant techniques to facilitate efficient purification thereof byIMAC. For this reason, IMAC has assumed a more important role inlarge-scale protein and/or peptide production. In addition, IMAC hasalso been used in purification of phosphorylated proteins and peptidesfrom tryptic protein digests. Such phosphorylated proteins and peptidescan subsequently be analysed by ESI/MS/MS to determine thephosphorylated sites therein.

Further, during the period when the IMAC was relatively new, use thereoffor purification of various compounds were suggested. For example,Porath et al (U.S. Pat. No. 4,677,027) disclosed in 1985 how biologicalmacromolecules and particles can be separated using a product consistingof a solid phase having immobilised metal ions on its surfacesubstituted via a metal chelate bond with a polymer. The envisagedbiomolecules are virus and cells, but polysaccharides, proteins and alsooligonucleotides are mentioned. However, since then, oligonucleotideshave due to more recent scientific findings found new applications, inturn necessitating novel modifications thereof.

One example of a more recently developed field, wherein oligonucleotidesare modified, is the antisense technology in drug discovery. Antisensedrugs work at the genetic level to interrupt the process by whichdisease-causing proteins are produced. This is possible, since proteinshave been shown to play a central role in virtually every aspect ofhuman metabolism. Almost all human diseases are the result ofinappropriate protein production, or a disordered protein performance.This is true of both host diseases, such as cancer, and infectiousdiseases, such as AIDS. Traditional drugs are designed to interactthroughout the body with protein molecules that support or causediseases. Antisense drugs are designed to inhibit the production ofdisease-causing proteins. They can be designed to treat a wide range ofdiseases including infectious, inflammatory and cardiovascular diseasesand cancer and have the potential to be more selective, and, as aresult, more effective and less toxic than traditional drugs. Themechanisms behind antisense technology have been widely described, seee.g. Uhlmann et al in Antisense Oligonucleotides: A New TherapeuticPrinciple, Chemical Reviews, Vol. 90, Number 4, June 1990. In brief, asis well known, during transcription of DNA into RNA, the twocomplementary strands of the DNA partly uncoil, whereby the strand knownas the sense strand separates from the strand known as the antisensestrand. The antisense strand is then used as a template for transcribingenzymes that assemble mRNA in the process known as transcription. ThemRNA then migrates into the cell, where ribosomes read the encodedinformation and string together amino acids to form a specific proteinin the process known as translation. Now, the antisense drugs arecomplementary strands of small segments of mRNA, and they can be eitherDNA or RNA. To create antisense drugs, nucleotides are linked togetherin short chains known as oligonucleotides. Each antisense drug isdesigned to bind a specific sequence of nucleotides in its mRNA targetto inhibit production of protein encoded by the target mRNA.

The linking together of oligonucleotides can be performed in any kind ofcommercially available automated solid-phase synthesiser for synthesisof oligonucleotides under cGMP conditions for clinical studies andcommercial drug supplies.) In such synthesis, the oligonucleotides,wherein one oxygen atom of the phosphate group of each base in thenative nucleic acid has been exchanged for a sulphur atom, are easilyproduced. However, an inherent problem in the synthesis of such thioatedoligonucleotides, herein-denoted antisense oligonucleotides, is the factthat it will be practically impossible to perform with a yield of 100%correctly phosphorothioated oligonucleotides. Instead, a yield in therange of about 70-75% is usually obtained. Accordingly, before anyantisense drug can be prepared thereof, the synthesised product willrequire a subsequent purification in order ensure a sufficient quality.

Reverse phase HPLC is commonly used for purification of antisenseoligonucleotides. However, use of high pressures is in general notconsidered to be advantageous conditions for this kind of process, sinceit put high demands on the equipment used and also makes the processdifficult, and consequently costly, to scale-up. In addition, theorganic solvents commonly used in this technology may be undesirable forsome applications.

Deshmukh et al (Deshmukh, R. R., Miller, J. E., De Leon, P., Leitch, W.E., Cole, D. L., and Sanghvi, Y. S. in “Process Development forPurification of Therapeutic Antisense Oligonucleotides by Anion-ExchangeChromatography”, Organic Process Research & Development 2000, 4,205-213) describes the development of an anion-exchange chromatographymethod for purification of phosphorothioate antisense oligonucleotides.More specifically, 20-mers which are antisense inhibitors of the celladhesion molecule ICAM-1 were synthesised and subsequently purified onan anion exchanger carrying quaternary arnmonium functional groups on apolystyrene-based matrix (Source 15 and Source Q 30, both from AmershamBiosciences AB, Uppsala, Sweden). The most advantageous resolution isobserved for the higher pH value tested for elution, which was pH 11.However, it has still to be shown whether or not a fully thioated 20-mercan be separated from a 20-mer, wherein one or more of the targetoxygens have not been substituted with sulphurs. Thus, the selectivityobtainable with ion exchange for purification of antisenseoligonucleotides is still not fully satisfactory. In addition, anotherdisadvantage is that such purification of antisense oligonucleotides byanion-exchange chromatography will also require a step of desaltingafterwards, which involves a further process step and consequently ahigher process cost in total.

Similarly, Deshmukh et al (Deshmukh, R. R., Warner, T. N., Hutchison,F., Murphy, M., Leitch, W. E., De Leon, P., Srivatsa, G. S., Cole, D.L., and Sanghvi, Y. S. in “Large-scale purification of antisenseoligonucleotides by high-performance membrane adsorber chromatography”,Journal of Chromatography A, 890 (2000) 179-192) have suggestedpurification of antisense oligonucleotides using strong anion exchangemembranes. However, like in the above described method, the selectivityobtainable is still not fully satisfactory (is this true, can we add anyother disadvantages/differences). In addition, use of membranes entailsa low capacity and hence large size membranes will be required for areasonably efficient process. Finally, this method will like theabove-discussed anion-exchange also require a step of desaltingafterwards.

WO 99/09045 (Somagenics, Inc.) relates to antisense and antigenetherapeutics with improved binding properties and methods for their use.More specifically, the invention relates to antisense and antigeneoligonucleotides capable of topologically linking to target nucleic acidin a manner that improves translation and transcription inhibitoryproperties. In one embodiment, phosphorothioate analogues of nucleicacids are disclosed, which have sulphur in place of non-bridging oxygensbonded to phosphorous in terminal or internucleotide phosphates. Thismodification is allegedly capable of a stronger binding tometallo-affinity chromatography media than the unmodified equivalents.However, there is no suggestion or guidance that metallo-affinitychromatography could be useful to separate oligonucleotides having avarying degree of thioation. Further, in another embodiment, theoligonucleotides have been platinated. Such platinated oligonucleotidesare easily separated from reaction mixtures by preparativeelectrophoresis, or alternatively by ion-exchange column chromatography.It is also suggested to use metallo-affinity chromatography onmercurated columns as a one-step method of purification of platinatedoligonucleotides, but this is a mere suggestion. Nothing in thisdocument provides any evidence that such purification would be efficientor even work, and the components of said “reaction mixture” are notdefined.

Thus, there is still a need of alternative procedures for thepurification of antisense oligonucleotides, especially of methodssensitive enough to separate antisense oligonucleotides of differentthioation degree from each other.

SUMMARY OF THE PRESENT INVENTION

One object of the present invention is to provide a method of isolatingantisense oligonucleotides from corresponding incorrectly synthesisedoligonucleotides and/or not fully thioated oligonucleotides in abiological solution. This can be achieved by the method as defined inthe claims.

A specific object of the invention is to provide a method of isolatingantisense oligonucleotides from a biological solution, which methodexhibits an improved selectivity as compared to the prior art methods.

Another object of the invention is to provide a method of isolatingantisense oligonucleotides from a biological solution, which methodreduces the need of organic solvents and/or high pressures as comparedto prior art methods.

A further object of the present invention is to provide a method ofisolating antisense oligonucleotides from a biological solution, whichmethod is easy to scale up and hence more cost-effective than the priorart methods.

Other objects and advantages of the present invention will appear fromthe detailed disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of a seven-base full-length, fully thioatedphosphorothioate (a); its monophosphodiester analogue (b) and a singledeletion sequence (c).

FIG. 2 shows IMAC using Fe³⁺ as metal ion as described in Example 1below and illustrates a comparison of elution of two differentoligonucleotides with the same sequence of bases.

FIG. 3 shows IMAC using Zr²⁺ as metal ion as described in Example 2below and illustrates a comparison of elution of two differentoligonucleotides with the same sequence of bases.

FIG. 4 shows an example of an efficient separation of a fully thioated(20S) oligonucleotide from an oligonucleotide with two phosphodiesterbonds (“2P”) using IMAC.

FIG. 5 shows a clear separation of a fully thioated (20S)oligonucleotide from an oligonucleotide with two phosphodiester bonds(“2P”) using IMAC, this time by step-wise elution.

DEFINITION

In this specification, the term “oligonucleotide” is used in itsconventional meaning, i.e. to mean a sequence of nucleotides, and theterm “polynucleotide” refers to a longer sequence of nucleotides thanthe oligonucleotide.

The term a “nucleotide” means a residue comprised of three parts, namelyan inorganic phosphate, a simple sugar and either a purine or apyrimidine base. In each nucleotide, the three parts are attached toeach other in the order-phosphate-sugar-base-. In an oligonucleotide,ester bonds link the sugar and phosphate components of adjacentnucleotide monomers. Since the sugar and the phosphate within anucleotide monomer are also linked via an ester bond, thesugar-phosphate-sugar linkage along the backbone of a poly- oroligonucleotide chain is known as a phosphodiester bond.

The term “chromatography” encompasses chromatographic separation methodsperformed in packed columns, in expanded or suspended beds and onmembranes.

The term “resin” refers to the solid phase used in chromatography, i.e.the adsorbent that captures the target species. A “resin” may beproduced in the form of porous or non-porous spherical or essentiallyspherical particles, beads, such as beads for expanded bed adsorption,and monoliths. Further, by providing the resin on a support, membranescan be provided, which are also useful for isolation of a species from aliquid. A resin is also known in this field as a matrix.

The term “adsorption” means herein the binding of a species to a ligandon a resin.

The term “eluent” is used herein in its conventional meaning i.e. for asolution capable of perturbing the interaction between the solid phase(resin) and product (target species) and promoting selectivedissociation of the product from the solid phase.

Consequently, the term “desorption” means to perturb the interaction asexplained above.

The term “buffer” or “buffered solution” refers to a mixture of acid andbase which when present in a solution reduces or modulates changes in pHthat would otherwise occur in the solution when acid or based is added.

The term “isolation” means herein a separation from other components andprovides a substantially pure target compound, such as a substantiallypure antisense oligonucleotide.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is a method of isolating fullythioated single stranded antisense oligonucleotides from a biologicalsolution, comprising the steps of contacting the biological solutionwith an immobilised metal ion adsorption chromatography (IMAC) resin toadsorb the antisense oligonucleotides to said resin and subsequentlycontacting the resin with an eluent under conditions that providedesorption of the antisense oligonucleotides from said resin, whereinthe fully thioated antisense oligonucleotides are separated fromincorrectly thioated antisense oligonucleotides in said solution. Thus,the present invention enables to purify the fully thioated singlestranded antisense oligonucleotides other components of a biologicalsolution, and hence allows to obtain said oligonucleotides in asubstantially pure form.

As is well known to those of skill in this field, during the synthesisof antisense oligonucleotides, besides oligonucleotides of an incorrectlength, the most prevalent contamination is oligonucleotides that havenot been fully thioated. Accordingly, the present invention fulfils animportant need in the production of antisense oligonucleotides fortherapeutical or other applications.

Thus, the present invention utilises for the first time to our knowledgethe interaction of a metal with the backbone phosphothioate group of anucleic acid in the purification of antisense oligonucleotides. Withoutwishing to limit the present invention to any specific interactions, itis also assumed that the nitrogen atoms of one or more of the basesadenine, guanine, uracil, cytosine and thymine of the oligonucleotidemay also be involved in this binding.

In the present context, it is to be understood that the term “fully”thioated means that in 100% of the phosphate backbone groups present ina corresponding native oligonucleotide, one of the non-bridging oxygenatoms in the phosphate backbone has been replaced by a sulphur atom.

The IMAC resin used in the present method can be any resin, such as theonce exemplified in the section “Background”. In brief, metal chelatinggroups include for example the iminodiacetic (IDA) group, thetris(carboxymethyl)-ethylenediamine (TED) group, the N-(hydroxyethyl)ethylenediaminetriacetic group, and derivatives such as the N-(methyl),and the N-(hydroxymethyl) IDA groups. These groups can be cross-linkedto the natural or synthetic polymeric support by standard aliphaticether linkages and reagents, such as bisoxirane, epichlorhydrin, and1,4-bis-(2,3-epoxypropoxy)butane. Examples of natural polymeric supportmaterials are e.g. agarose, alginate, carrageenan, gelatin etc.Synthetic polymers can be illustrated by styrene or derivatives,divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinylesters, vinyl amides etc, optionally cross-linked with any conventionalcross-linker, such as divinylbenzene, di- or polyfunctional(meth)acrylate esters, di- or polyfunctional (meth)acrylamides,triallylisocyanurate, divinylamides. For clarity, in this context, it isunderstood that an IMAC resin as used in the present method is comprisedof a support to which chelating groups have been attached, and chargedwith coordinating ions. Examples of suitable coordinating metal ions aree.g. Al, Ce, Cu, Co, Fe, In, Ga, Ge, Lu, Ni, Ru, Sb, Sc, Sn, Yc, Zn, Zr,Ta and Th ions. In one embodiment of the present invention, the metalion is Zr²⁺ or Fe³⁺. According to the present invention, this embodimentprovides a stronger bond to the phosphor of the bridge than to thecorresponding sulphur. IMAC resins are also commercially available, suchas HiTrap™ Chelating HP Columns and Chelating Sepharose™ Fast Flow, bothfrom Amersham Biosciences AB, Uppsala, Sweden.

In this context, it is understood that the term “resin” is used toencompass particles and beads as well as monoliths and membranes.

The desired antisense oligonucleotides can be separated from many kindsof components of the biological solution, such as proteins orincorrectly synthesised oligonucleotides, in large depending on thenature of the biological solution. Thus, in one embodiment, thebiological solution is provided from an automated synthesis of antisenseoligonucleotides. Hence, in this embodiment, the biological solution isa synthesis solution. In a similar embodiment, the biological solutionis a solution wherein the antisense oligonucleotides have beensynthesised using non-automated methods. Thus, synthesis can beperformed in solution according to well-known methods or in anycommercially available kind of equipment, such as a ÄKTA™ oligopilot(Amersham Biosciences AB, Uppsala, Sweden). In another embodiment, thebiological solution is serum, such as human serum, and the purpose ofthe method can then be to quantify the antisense oligonucleotidespresent therein. This embodiment can be part of treatment scheme,wherein it is desired to test the presence of drug i.e. antisenseoligonucleotide in the blood of the patient.

In one embodiment, the isolated single stranded (ss) antisenseoligonucleotides are of a size in the range of 10-30 bases, such as15-25 bases and more specifically 18-21 bases. In a specific embodiment,the antisense oligonucleotides are of a size in the range of about 18-20bases. In another embodiment, the antisense oligonucleotides arecomprised of up to about 25 bases, such as up to 20 bases. In yetanother embodiment, the antisense oligonucleotides are comprised of atleast 5 bases, such as at least about 10 bases. However, in thiscontext, since it is well-known that the kind of condition to be treatedusing the antisense technology will decide the nature, such as the basesequence and the size, of the antisense oligonucleotide, it isunderstood that the present invention also encompasses shorter or longeroligonucleotides as well, if they are useful in an antisensetechnology-based drug. Such drugs are useful in the treatment of bothhost diseases, such as cancer, and infectious diseases, as discussed infurther detail in the section “Background” above.

However, as also indicated in the background section above, thesynthesis of antisense oligonucleotides often results in part inincorrectly synthesised antisense oligonucleotides. The most commonimpurities in a biological solution that results from such synthesis aredeletion sequences, i.e. antisense oligonucleotides which are one ormore bases shorter than the desired product. Such deletedoligonucleotides can be described as (n−1) mers, (n−2) mers, (n−3) mersetc, wherein n denotes the number of nucleotides of the desiredfull-length product. Thus, in one embodiment of the present method, thefully thioated antisense oligonucleotides are separated from incorrectlysynthesised oligonucleotides. Other examples of incorrectly synthesisedoligonucleotides are addition sequences, i.e. antisense oligonucleotidesthat are longer than the desired products, and branched products.

Another example of undesired components in a biological solutionresulting from antisense oligonucleotide synthesis is incorrectlythioated sequence, i.e. not fully thioated oligonucleotides. Asmentioned above, these are one of the most commonly occurringcontaminations in a synthesis solution. The most prevalent form isoligonucleotides with one or two bonds without thioation. Thus, in oneembodiment of the present method, fully thioated antisenseoligonucleotides are separated from incorrectly thioated antisenseoligonucleotides containing 1 to 5, such as 1 or 2, bonds withoutthioation. Further examples of incorrectly thioated oligonucleotides arefor example 20-meric oligonucleotides wherein one phosphodiester grouphas not been correctly thioated, and hence oligonucleotide which areabout 95% thioated are separated from the fully thioated ones.Similarly, a 19-meric, 18-meric or 17-meric oligonucleotide wherein onebase has not been correctly thioated is thioated to about 94%.Accordingly, in one embodiment, the present fully thioated antisenseoligonucleotides are isolated from oligonucleotides that are thioated toabout 90%, such as about 94%, and preferably to about 95%. In anotherembodiment, the present fully thioated antisense oligonucleotides areisolated from oligonucleotides that are thioated to about 40%,preferably to about 60%, more preferably to about 80% and mostpreferably to about 90%.

In the prior art, when proteins and/or peptides have been isolated usingIMAC, conditions of neutral or close to neutral pH, such as about7.5-8.0, have been utilised. The present inventors unexpectedly foundthat when antisense oligonucleotides are isolated using IMAC, a lower pHis more favourable. Thus, in one embodiment of the present method, theconditions for adsorption are defined by a pH value below neutral. In aspecific embodiment, the pH is adapted to below about 7, such as about5. Thus, the pH of the biological solution at the contact with the resinmay be in a range of 0-7, 0-6 or 0-5. The pH is easily adjusted by theskilled person in this field by adding a suitable buffer or acid, suchas dilute acetic acid. In an advantageous embodiment, the pH is adjustedto about 5.0 and the buffer used is 15 mM sodium acetate. As is easilyrealised, since oligonucleotides are sensitive to extreme pH values,care should be taken not to adjust the pH in any way that can harm theantisense oligonucleotides.

The elution of the desired antisense oligonucleotides from the resin canbe performed according to standard methods using an increasing pH and/orphosphate gradient, for example using potassium phosphate. Anillustrative gradient is as used in the experimental part below, namelystarting from the pH used for adsorption, such as from 0.1% acetic acidto 0.5 M potassium phosphate. In an alternative embodiment, the gradientis from pH 3.0 to 0.2 M potassium phosphate. Other well-known salts andbuffers are also useful for the elution, and the skilled person caneasily set the appropriate conditions for elution. As the skilled personin this field will realise, the addition of salt will increase the ionicstrength, and hence the pH surrounding the antisense oligonucleotideswill change slightly. However, the pH in general during the adsorptionof the antisense oligonucleotides will still be lower than theconditions known for use of IMAC for protein separation.

In a specific embodiment, the present method in addition comprises asubsequent step of polishing the isolated antisense oligonucleotides.Such polishing is easily performed by the skilled person in this field,such as by gel filtration, detritylation precipitation, desalting,change of buffer etc.

Even though the examples shown below utilises a small lab scale, it isunderstood that the skilled person in this field can easily scale up thepresent method to s size useful in a production plant. Thus, oneadvantage with the present method is that it requires less expensivesolvents and equipment than e.g. the previously suggested reverse phasechromatography (RPC) method.

A second aspect of the present invention is an antisense oligonucleotideisolated by a method as defined above. Thus, the fully thioated singlestranded antisense oligonucleotides according to the invention areobtained in a purity of at least about 80%, more preferably at leastabout 90% and most preferably at least about 95%, such as close to 100%.

A third aspect of the present invention is the use of an immobilisedmetal affinity chromatography (IMAC) resin for isolation of antisenseoligonucleotides from corresponding oligonucleotides in a biologicalsolution. The IMAC resin can be as discussed in relation to the methodaccording to the invention, and the considerations discussed above mayalso apply to the present use.

Finally, the present invention also relates to a kit for purification offully thioated single stranded antisense oligonucleotides from abiological solution, which kit comprises a chromatography column packedwith an immobilised metal ion adsorption chromatography (IMAC) resin andwritten instructions for separation of said fully thioatedoligonucleotides from not fully thioated oligonucleotides. The presentkit may comprise a column of laboratory scale or a column of a sizesuitable for large-scale production of antisense oligonucleotides.Further, the kit may comprise buffer(s) suitable for elution andoptionally also for washing in a separate compartment(s).

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of a seven-base full-length, fully thioatedphosphorothioate (a); its monophosphodiester analogue (b) and a singledeletion sequence resulting in a (n−1) mer (c).

FIG. 2 shows IMAC using Fe³⁺ as metal ion as described in Example 1below and illustrates a comparison of elution of two differentoligonucleotides with the same sequence of bases. The X-axis shows theretention volume in ml, while the Y-axis shows the UV absorbance at 260nm in mAU. One of the oligonucleotides is fully thioated (denoted 20S inFIG. 2), while the other one is unmodified (denoted 20P in FIG. 2). Itappears clearly that the antisense oligonucleotide can be separated fromthe phosphodiester (non-modified) form of oligonucleotides, the thioatedform being eluted as a relatively narrow peak at 7.3 ml, before theunmodified form. The two small peaks eluted early in the chromatogramare presumably synthesis-related, and are caused by impurities in thesample that do not contain any phosphotioates or phosphodiester groups.

FIG. 3 shows IMAC using Zr²⁺ as metal ion as described in Example 2below and illustrates a comparison of elution of two differentoligonucleotides with the same sequence of bases. X- and Y-axis are asdescribed above. One of the oligonucleotides is fully thioated (denoted20S in FIG. 3), while the other one is unmodified (denoted 20P in FIG.3). It appears clearly that the antisense oligonucleotide can beseparated from the phosphodiester (non-modified) form ofoligonucleotides, the thioated form again being eluted as a relativelynarrow peak at about 9.4 ml, before the unmodified form. The two smallpeaks eluted early in the chromatogram are explained as above for FIG.2. A comparison of FIG. 2 and FIG. 3 reveals a stronger affinity of theoligonucleotides for the Zr-ion than the Fe-ion, however it is notedthat the conditions used have not been optimised.

FIG. 4 shows an example of an efficient separation of twooligonucleotides having the same sequence, as described in detail inexample 3. More specifically, this drawing shows that it is quitepossible to separate a fully thioated (20S) oligonucleotide from anoligonucleotide with two phosphodiester bonds (“2P”) using IMAC. Thepeaks are clearly separated in the chromatogram. For elution, a 10 CVlinear gradient from 15 mM sodium acetate to 0.2 M potassium phosphatewas used.

FIG. 5 shows again a clear separation of two oligonucleotides having thesame sequence as described in example 4. More specifically, this drawingshows a complete, baseline resolution of the peaks corresponding to afully thioated oligonucleotide (20S) from that of an oligonucleotidewith two phosphodiester bonds (“2P”). Thus, the present invention showsthat it is possible to separate a fully thioated oligonucleotide from anoligonucleotide with two phosphodiester bonds using IMAC and step-wiseelution. For elution, a step gradient was used: Step 1 was at 0.1 Mpotassium phosphate and step two at 0.2 M potassium phosphate. Theresults obtained in examples 3 and 4 as illustrated in FIGS. 4 and 5provide evidence that supports an essential importance of thephosphonate groups in the present binding, not the bases. Thus, thiscontradicts what was indicated in the above discussed WO 99/09045, whereit was stated that a higher degree of thioation would yield a strongerbinding to an IMAC resin.

EXPERIMENTAL PART

The present examples are provided for illustrative purposes only andshould not be construed as limiting the scope of the present inventionas defined by the appended claims. All references given below andelsewhere in the present specification are hereby included herein byreference.

Example 1 Purification of Single Stranded Antisense Oligonucleotides byIMAC Using Fe³⁺ as Metal Ion

The oligonucleotides used in this study were 20-mers with the sequenceGCC CAA GCT GGC ATC CGT CA (SEQ ID NO:1). Two different oligonucleotidewere used, one fully thioated and one without any modification(phosphodiester form).

For the study was used a small IMAC column with IDA chemistry, theHITRAP™ Chelating HP column (1 ml volume) (available from AmershamBiosciences AB, Uppsala, Sweden, Prod # 17-0408-01).

The solvents/buffer used in this example are for IMAC rather unusual. Asbinding “buffer” a solution of 0.1% acetic acid in water was used. Theelution was achieved with a 10 Column Volume linear gradient from 0.1%acetic acid in water to 0.05 M potassium phosphate. However, it is notedthat these conditions were not optimised, neither for binding(adsorption) nor for elution.

Flow rate of the eluent was 1 ml/min and detection was made with UV at260 nm.

Thus, the Fe³⁺ was tested and found useful as a metal ion in the methodaccording to the invention. The results of this example are as shown inFIG. 2.

Example 2 Purification of Single Stranded Antisense Oligonucleotides byIMAC Using Zr²⁺ as Metal Ion

The oligonucleotides used in this study were the 20-mers described inExample 1 above.

For the study was used a small IMAC column with IDA chemistry, theHITRAP™ Chelating HP column (1 ml volume) (available from AmershamBiosciences AB, Uppsala, Sweden, Prod # 17-0408-01).

In this example, the binding “buffer” was like in Example 1 a solutionof 0.1% acetic acid in water. The elution was achieved herein with a 10Column Volume linear gradient from 0.1% acetic acid in water to 0.2 Mpotassium phosphate. However, it is noted that these conditions were notoptimised either.

Flow rate of the eluent was 1 ml/min and detection was made with UV at260 nm.

Thus, Zr²⁺ was tested and found useful as a metal ion in the methodaccording to the invention. The results of this example are shown inFIG. 3.

Example 3 Purification of Synthetic (Antisense) Oligonucleotides fromNot Fully Thioated Oligonucleotides by IMAC, Elution by Linear Gradient

This example shows the method according to the present invention iscapable of separating fully thioated oligonucleotides from just partlythioated oligonucleotides.

The oligonucleotides used in this study were 20-mers with the sequenceGCC CAA GCT GGC ATC CGT CA (SEQ ID NO:1). Two different oligonucleotideswere used, one fully thioated and one with two of the bonds withoutmodification (phosphodiester form). The phosphodiester bonds were atposition 10 and 15 (defined from the 5′ end), respectively.

For the study was used a small IMAC column with IDA chemistry, theHITRAP™ Chelating HP column (1 ml volume) (Amersham Biosciences,Uppsala, Sweden, Prod # 17-0408-01). Zr²⁺ was the metal ion studied.

The solvents and buffers used herein are for IMAC rather unusual. Asbinding buffer, 15 mM sodium acetate and pH is 5.0 was used. The elutionwas achieved by potassium phosphate, 0.2 M, pH 6.5. The flow rate was 1ml/min and detection was made with UV at 260 nm.

The results are shown in FIG. 4, which also provides a comparisonbetween the fully thioated (20S) and the oligonucleotide with twophosphodiester bonds (“2P”).

Example 4 Purification of Synthetic (Antisense) Oligonucleotides fromNot Fully Thioated Oligonucleotides by IMAC, Elution by Step Gradient

This is a second example that illustrates how the method according tothe present invention is capable of separating fully thioatedoligonucleotides from partly thioated oligonucleotides. The startingmaterials and instruments were as in Example 3 above, the buffer is 15mM sodium acetate, pH 5.0, and in this example the elution is made by astep gradient. The first step is 2 CV at 0.1 M potassium phosphate andthe second step is with 2 CV at 0.2 M potassium phosphate. The resultsare provided in FIG. 5, which shows a separation of a mixture of twooligonucleotides, a fully thioated (20S) and an oligonucleotide with twophosphodiester bonds (“2P”).

1. A method of isolating fully thioated single stranded antisenseoligonucleotides from a biological solution, which method comprises thesteps of contacting the biological solution with an immobilised metalion adsorption chromatography (IMAC) resin to adsorb antisenseoligonucleotides to said resin and subsequently contacting the resinwith an eluent under conditions that provide desorption of the antisenseoligonucleotides from said resin, wherein the fully thioated antisenseoligonucleotides are separated from incorrectly synthesised and/orincorrectly thioated antisense oligonucleotides in said solution;further wherein the metal ion is Zr²⁺ or Fe³⁺.
 2. The method of claim 1,wherein the biological solution is a synthesis reaction of antisenseoligonucleotides.
 3. The method of claim 1, further wherein fullythioated antisense oligonucleotides are separated from incorrectlysynthesised oligonucleotides.
 4. The method of claim 1, wherein fullythioated antisense oligonucleotides are separated from incorrectlythioated antisense oligonucleotides containing 1-5 bonds withoutthioation.
 5. The method of claim 1, wherein the antisenseoligonucleotides are of a size in the range of 5-30 base pairs.
 6. Themethod of claim 1, wherein the pH of the biological solution is belowabout 7 during the adsorption of antisense oligonucleotides.
 7. Themethod of claim 1, which in addition comprises a subsequent step ofpolishing the isolated antisense oligonucleotides.